Genomic imprinting refers to an epigenetic marking of genes that results in parent-of-origin dependent, monoallelic expression. Imprinted genes have critical roles in embryonic growth and behavior; many also function as cancer susceptibility loci in somatic cells because their functionally haploid state makes them vulnerable to inactivation or overexpression. This heightened susceptibility is exacerbated by positional clustering of imprinted genes under the regional control of imprinting centers that, when disrupted genetically or epigenetically, lead to multigenetic phenotypic abnormalities including cancer. Imprinted genes may also mechanistically link nutritional perturbations like methylation deficiency directly to the etiology of cancer because their cis-acting regulatory elements are epigenetically labile. We propose to use phylogenetic comparisons of orthologous sequences from members of the three extant mammalian orders, Prototheria, Metatheria and Eutheria to characterize the evolution of imprinted domain structures and define the fundamental cis-acting imprint regulatory elements that epigenetically distinguish the parental alleles and also constitute targets for epigenetic dysregulation. The overall hypothesis of this grant application is that gene promoter silencing represents the primordial imprint mechanism, and that ongoing interparental genetic conflict has led to increased regulatory complexity for some imprinted domains, as the maternal and paternal genomes evolved counteracting strategies to overcome gene repression. Imprinting mechanisms are therefore postulated to be less complex in more ancestral mammals. To test this novel hypothesis we recently produced bacterial artificial chromosome (BAC) libraries from the imprinted opossum (Didelphus virginiana) and the non-imprinted platypus (Ornithorhynchus anatinus) to examine imprinted domains containing genes involved in cancer that are regulated by three different imprinting mechanisms. Maternal repression of expression will be modeled by NNAT (Neuronatin); paternal repression of expression with antisense transcripts will be modeled by M6P/IGF2R, and reciprocal parental repression of juxtapositioned genes by an intervening imprint center will be modeled by the IGF2/H19 and DLK1/MEG3 imprinted domains. We will test the functional relevance of novel regulatory elements identified in these comparisons by determining if they can direct acquisition of imprinting in transgenic mice. The successful completion of these comparative phylogenetic studies will significantly enhance our understanding of the evolution of this unique mammalian form of gene regulation. These studies will also be critical for identifying novel imprinted genes, and characterizing the less well-defined imprinted domains known to harbor genetic and/or epigenetic mutations mechanistically involved in cancer and neurogenetic disorders, such as schizophrenia, bipolar disease and autism.